Lantibiotics are ribosomally synthesized peptides that undergo posttranslational modifications to their mature, antimicrobial form. They are characterized by the unique amino acids lanthionine and methyllanthionine, introduced by means of dehydration of Ser͞Thr residues followed by reaction of the resulting dehydro amino acids with cysteines to form thioether linkages. Two-component lantibiotics use two peptides that are each posttranslationally modified to yield two functionally distinct products that act in synergy to provide bactericidal activity. By using genetic data instead of isolation, a two-component lantibiotic, haloduracin, was identified in the genome of the Gram-positive alkaliphilic bacterium Bacillus halodurans C-125. We show that heterologously expressed and purified precursor peptides HalA1 and HalA2 are processed by the purified modification enzymes HalM1 and HalM2 in an in vitro reconstitution of the biosynthesis of a two-component lantibiotic. The activity of each HalM enzyme is substratespecific, and the assay products exhibit antimicrobial activity after removal of their leader sequences at an engineered Factor Xa cleavage site, indicating that correct thioether formation has occurred. Haloduracin's biological activity depends on the presence of both modified peptides. The structures of the two mature haloduracin peptides Hal␣ and Hal␤ were investigated, indicating that they have similarities as well as some distinct differences compared with other two-component lantibiotics.lanthionine ͉ dehydroalanine ͉ antibiotic

Modification of the phosphate groups of lipid A with 4-amino-4-deoxy-L-arabinose (L-Ara4N Lipopolysaccharide (LPS)1 is an immunogenic glycolipid that constitutes most of the outer leaflet of the outer membrane of Gram-negative bacteria (1-4). LPS consists of three domains, which are the O-antigen, the core oligosaccharide, and the lipid A moiety (1-4). The O-antigen functions as a protective barrier, whereas the core sugars maintain outer membrane integrity and provide an attachment site for the O-antigen (1-4). Lipid A is the hydrophobic membrane anchor of LPS, and it is the active (endotoxin) component of LPS, accounting for many of the pathophysiological effects associated with Gram-negative sepsis (5-7).The Kdo 2 -lipid A portion of LPS is sufficient to support growth in Escherichia coli and Salmonella typhimurium (2). Covalent modifications to Kdo 2 -lipid A can be induced by environmental stimuli, such as low Mg 2ϩ concentrations or low pH (8 -10). As shown in Fig. 1 for S. typhimurium, these modifications include the incorporation of palmitate (11, 12), the addition of phosphoethanolamine (pEtN) (13-15), and/or the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) moieties (13,16,17). The modification of at least one phosphate residue with L-Ara4N is required for maintaining resistance to certain cationic antimicrobial peptides of the innate immune system and to the antibiotic polymyxin (18,19). Resistance is due, in part, to the neutralization of the negative charges of lipid A by L-Ara4N, reducing the affinity of lipid A for cationic substances (20) and preventing these anti-microbial compounds from penetrating the outer membrane.The addition of pEtN and L-Ara4N groups to lipid A is controlled by the PmrA/PmrB two-component regulatory system, which is activated by low pH, high Fe 3ϩ levels, or indirectly, by low concentrations of Mg 2ϩ via the PhoP/PhoQ system through the action of PmrD (9). Activated PmrA stimulates transcription at the pmrCAB, pmrE(ugd) , and pmrHFIJKLM loci (19,21). Constitutive pmrA (pmrA c ) mutants of E. coli and S. typhimurium are polymyxin-resistant, and they modify their lipid A with L-Ara4N and pEtN groups under all growth conditions (17,18,22). Inactivation of either pmrE or the genes in the pmrHFIJKLM operon results in complete loss of polymyxin resistance and of L-Ara4N-modified lipid A in pmrA c bacterial cells (19,21,23). Similarly, pmrA c mutants harboring a nonpolar disruption of the pmrC(eptA) gene are unable * This research was supported by National Institutes of Health Grant GM-51310 (to C. R. H. R.). The Duke University NMR Center is partially supported by P30-CA-14236. NMR instrumentation in the Center was funded by the National Science Foundation, the National Institutes of Health, the North Carolina Biotechnology Center, and Duke University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fac...

The zinc-dependent enzyme LpxC catalyzes the deacetylation of UDP-3-O-acyl-GlcNAc, the first committed step of lipid A biosynthesis. Lipid A is an essential component of the outer membranes of most Gram-negative bacteria, including Escherichia coli, Salmonella enterica and Pseudomonas aeruginosa, making LpxC an attractive target for antibiotic design. The inhibition of LpxC by a novel N-aroyl-L-threonine hydroxamic acid (CHIR-090) from a recent patent application (International Patent WO 2004/062601 A2 to Chiron and the University of Washington) is reported here. CHIR-090 possesses remarkable antibiotic activity against both E. coli and P. aeruginosa, comparable to that of ciprofloxacin. The biological activity of CHIR-090 is explained by its inhibition of diverse LpxC orthologs at low nM concentrations, including that of Aquifex aeolicus, for which structural information is available. The inhibition of A. aeolicus LpxC by CHIR-090 occurs in two steps. The first step is rapid and reversible, with a K i of 1.0 -1.7 nM, depending on the method of assay. The second step involves the conversion of the EI complex with a half-life of about a minute to a tightly bound form. The second step is functionally irreversible but does not result in the covalent modification of the enzyme, as judged by electrospray-ionization mass spectrometry. CHIR-090 is the first example of a slow, tight-binding inhibitor for LpxC, and may be the prototype for a new generation of LpxC inhibitors with therapeutic applicability.The emergence of multi-drug resistant bacterial pathogens is a growing public health concern (1). Human and animal pathogens are developing resistance to every major class of commercial antibiotic, both natural and synthetic. New antibiotics directed against previously unexploited bacterial targets are urgently needed (2-4).Zinc-dependent hydrolases are a well-studied class of proteins, many of which have set successful precedents for mechanism-based inhibitor design (5-7). Several bacterial metalloamidases have been identified as potential antibiotic targets (7). Among them is LpxC, a zincdependent, cytoplasmic deacetylase involved in the biosynthesis of the lipid A component of lipopolysaccharide (Scheme 1) (8-11).LpxC removes the acetate group from the nitrogen atom at the glucosamine 2 position of UDP-3-O-acyl-N-acetylglucosamine (Scheme 1) (12,13). This reaction is the first committed step of lipid A biosynthesis (14) and is essential for bacterial growth (12,13 Despite its unique substrate specificity and sequence, LpxC does share some mechanistic features with other important metallo-amidases. As in thermolysin (18), angiotensinconverting enzyme (5,19), the matrix metallo-proteinases (6), and peptide N-deformylase (20), a single transition metal ion is required for LpxC catalytic activity (9) and a glutamate side chain in the LpxC active site is thought to activate the Zn 2+ -bound water (21). Selective chelation of the LpxC active site Zn 2+ ion by certain small molecules containing hydroxamic acid group...